SAT-LB56 Undernutrition Reduces Transcript Abundance of Kisspeptin and Neurokinin B in Young Male Sheep

Abstract

Proper energy balance is important to ensure reproductive success. Chronic nutrient restriction is known to suppress hypothalamic-pituitary function, but the central mechanisms whereby undernutrition inhibits GnRH/LH secretion remain largely unknown. KNDy neurons, which co-express kisspeptin, neurokinin B (NKB), and dynorphin, form a unique population of cells in the arcuate nucleus (ARC) of the hypothalamus and play a critical role in GnRH/LH pulse generation. Based on recent evidence from our lab that chronic feed restriction reduces kisspeptin and NKB protein expression in young male sheep, we hypothesized that nutrient restriction would inhibit mRNA abundance for kisspeptin and NKB in the same animals. Fourteen wethers were placed into a fed to maintain body weight group (n=6; Fed) or a feed-restricted to lose 15-20% of pre-study body weight group (FR; n=8). Weekly blood samples (every 12 minutes for 4.5 hours) were taken via jugular venipuncture and plasma was stored at -20°C until the time of radioimmunoassay. Weekly body weights were recorded and feed amounts were adjusted to achieve desired body weights. At Week 13, animals were euthanized following blood collection, brain tissue was perfused with 4% paraformaldehyde, and tissue containing the hypothalamus was collected. Following submersion in 20% sucrose for at least four weeks, hypothalamic blocks were sectioned at 50 µm on a freezing microtome, and stored in a cryopreservative solution until processing. At Week 13, the average percent change in body weight was clearly evident (Fed, 6.79 + 3.4% vs FR, -19.82 ± 1.6%), and mean LH was significantly lower in FR wethers (13.41 + 3.7 ng/ml) compared to Fed controls (26.43 + 2.5 ng/ml). To assess changes in mRNA abundance, we used a relatively new in situ hybridization technique, RNAscope, to quantify mRNA for kisspeptin and NKB in the ARC with probes that were ovine-specific. Results showed that feed restriction reduced the number of kisspeptin mRNA-expressing cells (Fed, 231.2 + 14.4 vs FR, 100.3 + 35.9) and NKB mRNA-expressing cells (Fed, 192.7 + 18.4 vs FR, 97.3 + 21.7) per hemi-section. Furthermore, analysis of kisspeptin and NKB co-expressing cells (30 cells/animal) revealed that feed restriction significantly reduced the average mRNA integrated density for NKB, but not kisspeptin, compared to Fed controls. Together, these findings further support a role for kisspeptin and NKB in the central mechanism governing GnRH/LH secretion during undernutrition in male sheep.